The aim of the study was to evaluate the prevalence of anti-Chlamydia trachomatis (C. trachomatis) antibodies in serum and expressed prostatic secretion (EPS) in chronic prostatitis. Thirty-six patients with chronic prostatitis were examined. The presence of C. trachomatis was determined in urethral smears and EPS. Specific antibodies were determined in the serum (IgM, IgA, IgG) and in the EPS (IgA, IgG). In the direct diagnosis of chlamydial infection, the direct immunofluorescence method and the ligase chain reaction were employed, and for the serological diagnosis, the immunoenzymatic method. C. trachomatis infection was detected in the urethra of 3 (8.3%) patients and in the prostatic gland of 3 (8.3%) patients; only one of these patients was found to have C. trachomatis in both the urethra and the EPS. In the control group, C. trachomatis was detected in the urethra of 1/50 (2%) of the men, but the EPS of all of them was free of C. trachomatis. Specific IgM antibodies were found in 7 (19.4%), IgA in 9 (25%), and IgG in 18 (50%) of the patients' serum, whereas IgAs were detected in 12 (33.3%) and IgGs in 13 (36.1%) of the patients' EPS. In the control group, anti-C. trachomatis antibodies of the IgG were detected in the serum of 2/35 (5.7%) of the men, whereas in the EPS neither IgA nor IgG antibodies were detected in any of these patients. Serological tests of the serum and EPS are useful as a complementary method in the diagnosis of chronic prostatitis.